DNA methyltransferase 1 mutations and mitochondrial pathology: … · 2017-04-13 · Maresca et al. DNMT1 and mtDNA methylation comprise two parts: a C-terminal catalytic portion

REVIEWpublished: 12 March 2015

doi: 10.3389/fgene.2015.00090

Edited by:Babajan Banganapalli, King Abdulaziz

University, Saudi Arabia

Reviewed by:Khalid Moghem A. L. Harbi, Taibah

University, Saudi ArabiaXusheng Wang, St. Jude Children’s

Research Hospital, USA

*Correspondence:Claudia Zanna, Department ofPharmacy and Biotechnology,

University of Bologna, Via Irnerio 42,40126 Bologna, Italy

[email protected]

Specialty section:This article was submitted to Genetic

Disorders, a section of the journalFrontiers in Genetics

Received: 30 December 2014Accepted: 19 February 2015

Published: 12 March 2015

Citation:Maresca A, Zaffagnini M, Caporali L,

Carelli V and Zanna C (2015) DNAmethyltransferase 1 mutations andmitochondrial pathology: is mtDNA

methylated?Front. Genet. 6:90.

doi: 10.3389/fgene.2015.00090

DNA methyltransferase 1 mutationsand mitochondrial pathology: ismtDNA methylated?Alessandra Maresca1, Mirko Zaffagnini 2, Leonardo Caporali 3, Valerio Carelli 1,3 andClaudia Zanna 2*

1 Unit of Neurology, Department of Biomedical and NeuroMotor Sciences, University of Bologna, Bologna, Italy,2 Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy, 3 IRCCS Institute of NeurologicalSciences of Bologna, Bellaria Hospital, Bologna, Italy

Autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) andHereditary sensory neuropathy with dementia and hearing loss (HSN1E) are two rare,overlapping neurodegenerative syndromes that have been recently linked to allelicdominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5)-methyltransferase 1 (DNMT1). DNMT1 is the enzyme responsible for maintaining thenuclear genome methylation patterns during the DNA replication and repair, thusregulating gene expression. The mutations responsible for ADCA-DN and HSN1E affectthe replication foci targeting sequence domain, which regulates DNMT1 binding tochromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNAmethylation pattern with predictable downstream consequences on gene expression.Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical ofmitochondrial diseases, such as optic atrophy, peripheral neuropathy, and deafness,and some biochemical evidence of mitochondrial dysfunction. The recent discovery of amitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA(mtDNA) suggests that DNMT1 mutations may directly affect mtDNA and mitochondrialphysiology. On the basis of this latter finding the link between DNMT1 abnormalactivity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive,however, mtDNA methylation remains highly debated. In the last years several groupsdemonstrated the presence of 5-methylcytosine in mtDNA by different approaches,but, on the other end, the opposite evidence that mtDNA is not methylated has alsobeen published. Since over 1500 mitochondrial proteins are encoded by the nucleargenome, the altered methylation of these genes may well have a critical role in leadingto the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many openquestions still remain unanswered, such as why mtDNA should be methylated, and howthis process is regulated and executed?

Keywords: DNMT1 mutations, mtDNA methylation, ADCA-DN, HSN1E, mitochondrial dysfunction

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Maresca et al. DNMT1 and mtDNA methylation

DNA Methylation

Several epigenetic signals participate in cell specific gene expres-sion, including DNA methylation and demethylation, post-translational modifications of histone proteins (i.e., acetylation,methylation, phosphorylation, and ubiquitination), incorpora-tion of histone variants and gene regulation by non-coding RNAs(Rivera and Ren, 2013; Kanherkar et al., 2014a). DNA methyla-tion, which occurs in all prokaryotic and eukaryotic organisms,with rare exception for yeast, roundworm, and fruit fly, is a keyepigenetic process involved in the regulation of gene expres-sion (Lande-Diner et al., 2007; Lee et al., 2010) and parentalimprinting (Sha, 2008; Ishida and Moore, 2013), in chromo-some X inactivation (Straub and Becker, 2007) as well as in thedevelopment of the immune system (Cedar and Bergman, 2011;Kondilis-Mangum and Wade, 2013; Li et al., 2013) and in cellularreprogramming (Reik, 2007; Krishnakumar and Blelloch, 2013;Papp and Plath, 2013; Kanherkar et al., 2014b). Furthermore,it is engaged in the maintenance of the genome integritythrough protection against endogenous retroviruses and trans-posons (Howard et al., 2008). In humans aberrant DNA methy-lation patterns are associated with several diseases, includ-ing various cancers (Jones and Baylin, 2007; Esteller, 2008;Iacobuzio-Donahue, 2009), immune system disorders (Feinberg,2007) and neurodegeneration (Jakovcevski and Akbarian, 2012;Qureshi and Mehler, 2013).

Classical Model of DNA MethylationIn prokaryotes, DNA methylation occurs on both cytosine andadenine bases and is one of the host restriction systems todistinguish self and non-self DNA (Jeltsch, 2006). In mam-mals, following the classical model of DNA methylation, ittakes place in the cytosine residues at their C5 positions, pri-marily in the CG dinucleotides (CpG), acting mainly as arepressive tag to silence chromatin and inhibit transcription.

In the human genome there are 56 million CG sites, about60–80% of which are methylated, corresponding to 4–6% ofall cytosines (Laurent et al., 2010). Methylation levels and pat-terns are cell and tissue specific. In mammalian genomes, theCpG are poorly represented compared to other dinucleotides,because of the higher mutagenic property of the 5-methyl-cytosine base compared to the unmethylated one (Pfeifer et al.,2000). The irregular CpG distribution is reflected by theirdepletion in intergenic and intragenic sequences, whereas theirpresence is less suppressed in repetitive DNA (such as trans-posons and retroviruses) and CpG islands, regions of at least550 bp, with a ratio of observed CpG/expected CpG higherthan 0.65 (Takai and Jones, 2004). Around 70% of human genespromoter regions present the CpG islands and an inversecorrelation between CpG islands density and the promotermethylation status exists. Moreover, active genes usually showhypomethylation at the transcriptional start site (TSS) andhigh levels of methylation in the gene body, which is sup-posed to block aberrant transcription initiation inside the gene,avoiding the production of truncated mRNAs and proteins.The splicing sites are regions characterized by a change inDNA methylation, since exons have higher methylation thanintrons, suggesting a strong implication of DNA methylation inthe splicing process (Laurent et al., 2010; Chatterjee and Vinson,2012).

DNA Methyltransferase EnzymesDNA methylation is catalyzed by a group of enzymes calledDNA (cytosine-5)-methyltransferases (DNMTs) that transfera methyl group from a cofactor molecule S-adenosyl-L-methionine (AdoMet or SAM) to the C5 position of thecytosine residues to generate 5-methylcytosine (5 mC) andS-adenosyl-L-homocysteine (AdoHcy, SAH; Figure 1A).The mammalian DNMT family includes four members:DNMT1, DNMT3A, DNMT3B, and DNMT3L. These enzymes

FIGURE 1 | Schematic representation of DNA methylation. (A) DNA(cytosine-5)-methyltransferases (DNMTs) transfer the methyl group at the DNAcytosine ring carbon C5 by using S-adenosyl methionine (SAM) as methyl

donor. S-adenosyl homocysteine (SAH) is the cofactor product. (B) DNAmethylation: classical model. Maintenance versus de novo methylation. Figuremodified by Cheng and Blumenthal (2008).






comprise two parts: a C-terminal catalytic portion and alarge N-terminal region of variable size containing reg-ulatory domains involved in the interaction with DNA,chromatin, and other proteins (Figure 2). Furthermore, theN-terminal region contains 621 amino acids required fordiscriminating between hemi-methylated and unmethy-lated DNA. The C-terminal catalytic domain, being highlyconserved between eukaryotes and prokaryotes, is com-posed by 500 amino acids and harbors the active centerof the enzyme, which contains amino acids motifs char-acteristic of the cytosine-C5 methyltransferases, called the“AdoMet-dependent MTase fold” (Jurkowska et al., 2011a).Motifs I and X of this domain are involved in cofactorbinding whereas motifs IV, VI, and VIII have a catalyticfunction. The non-conserved region between motifs VIIIand IX is the target recognition domain (TRD), crucial forDNA recognition and specificity (Cheng, 1995; Jeltsch, 2002;Cheng and Blumenthal, 2008). Considering the high variabilityof the N-terminus of DNTMs, a detailed description of this

region for each DNMT isoforms will be provided in the followingparagraphs.

The initial methylation pattern is established by de novoDNMTs (DNMT3 family in mammals). This pattern is perpet-uated for the rest of the life (with small tissue-specific changes)by a mechanism first proposed by Holliday and Pugh (1975) andRiggs (1975).

Each round of DNA replication produces hemimethylatedDNA with the methylation marks in the parental stand andthe new synthesized daughter unmethylated strand (Figure 1B).The maintenance DNA methyltransferase (DNMT1 in mam-mals), showing a preference for hemimethylated sites, copies theexisting methylation pattern (Jones and Liang, 2009; Denis et al.,2011).

DNMT3 FamilyDuring the embryogenesis the DNMT3 family establishesthe initial CpG methylation pattern (Chen and Li, 2006;Jurkowska et al., 2011a). The DNMT3 family includes two active

FIGURE 2 | A schematic representation of the domain structure ofhuman DNMT isoforms. For human DNMT1, DNA methyltransferaseassociated protein (DMAP), replication foci targeting sequence (RFTS), CXXC,bromo-adjacent homology (nBAH/cBAH), and catalytic domains arerepresented by cyan, blue, orange, violet, and yellow bars, respectively. Forhuman DNMT2, catalytic domain is represented by yellow bar. For humanDNMT3A, DNMT3B, and DNMT3L, PWWP, ADD, and catalytic domains arerepresented by green, red, and yellow bars, respectively. In the catalytic domain(yellow bar), the conserved C5 DNA MTase motifs in the C-terminal part of eachDNMT isoforms are labeled. The interdomain linker regions are shown as lightgray bars. Numbers in parentheses indicate the length of each protein. The

crystal structures of the human DNMT1 RFTS domain (residues 351–597, PDBID: 3EPZ; Syeda et al., 2011), human DNMT1 CXXC, nBAH/cBAH, and catalyticdomains bound to DNA-containing unmethylated CpG sites (residues646–1600, PDB ID: 3PTA; Song et al., 2011), human DNMT2 catalytic domain(residues 1–188 and 248–391, PDB ID: 1G55; Dong et al., 2001), humanDNMT3A PWWP (residues 275–427, PDB ID: 3LLR), and ADD domains(residues 476–614, PDB ID: 3A1A; Otani et al., 2009; Wu et al., 2011,respectively), human DNMT3B PWWP domain (residues 293–442; PDB ID:3QKJ; Wu et al., 2011), human DNMT3L ADD domain (residues 34–277 and279–380, PDB ID: 2PV0; Ooi et al., 2007), are shown. For further details see“Text.”






de novo DNMTs, DNMT3A, and DNMT3B, without significantpreference between hemimethylated and unmethylated DNA,and one catalytically inactive regulatory factor, the DNMT3-Likeprotein (DNMT3L; Bestor, 2000; Jurkowska et al., 2011a).

Knockout of DNMT3A or DNMT3B is lethal, being bothessential for embryonic development in mice. Mouse DNMT3Bknockout embryos die in utero, whereas the DNMT3A knock-out animals die shortly after birth (Okano et al., 1999). DNMT3Lknockout mice are viable, but male are sterile, failing to pro-duce mature sperm (Hata et al., 2002). Mutations in the humanDNMT3B gene are associated with a rare autosomal disease,called ICF (Immunodeficiency, Centromere instability, Facialabnormalities) syndrome, which is accompanied by hypomethy-lation of classical satellites of the pericentromeric regions ofchromosomes 1, 9, and 16, probably due to reduction of enzymecatalytic efficiency or alteration of its localization (Xu et al.,1999; Weemaes et al., 2013). De novo mutations in DNMT3Ahave also been identified in patients with overgrowth disorders(Tatton-Brown et al., 2014). Despite high sequence homologyand similar biochemical properties, DNMT3A and 3B show par-tially non-overlapping biological functions with DNMT3A beinginvolved in the setting of parental imprints and DNMT3B inthe methylation of pericentromeric repeats (Okano et al., 1999;Kaneda et al., 2004).

From the structural point of view, the DNMT3 enzymes con-tain a N-terminal variable portion (around 280 and 220 aminoacids for DNMT3A and DNMT3B, respectively), followed by twoconserved regions: the regulatory PWWP and ADD domains andC-terminal catalytic portion. DNMT3L lacks the PWWP domainas well as the DNMTs motifs IX and X and all the important cat-alytic residues in its C-terminal portion (Figure 2). The PWWPis a variable module of 100–150 amino acids characterized by thepresence of a strictly conserved proline-tryptophan-tryptophan-proline motif, also found in chromatin associated eukaryotic pro-tein from yeast to mammals. It comprises a globular domain witha five-stranded β-barrel followed by a five-helix bundle (Figure 2;Qiu et al., 2002; Chen et al., 2004). In vitro experiments showedthat the PWWP domain of DNMT3B mediate DNA binding,but it is not required for CpG methylating activity (Qiu et al.,2002). Subsequently, it has been demonstrated that it exhibitsa dual function: binding to both DNA and methylated lysineon histone proteins (Chen et al., 2004; Dhayalan et al., 2010;Wu et al., 2011). The ADD domain (ATRX-DNMT3-DNMT3L),also known as PHD domain (plant homodomain), is present inall the proteins belonging to DNMT3 family and in the ATRX(alpha thalassemia/mental retardation syndrome X-linked) pro-tein. This domain is a cysteine-rich module that binds zincions (comprising six CXXC motifs; Figure 2) and constitutes aplatform for various protein–protein interactions. It has beenreported that DNMT3L, which only owns this characteristicmotif, recognizes specifically the unmethylated lysine 4 of his-tone H3 through its ADD domain and induces de novo DNAmethylation by recruitment or activation of DNMT3A, thanks toan interaction between its C-terminal domain and the catalyticportion of DNMT3A. These data support the idea that DNMT3Lhas the dual function of binding the unmethylated histone tailand activating DNA methyltransferase (Jia et al., 2007; Ooi et al.,

2007). Nevertheless, the ADD domains of DNMT3A and 3Bshare considerable homology with DNMT3L and it has beenreported a direct binding of the DNMT3A and DNMT3B ADDdomains to H3 tails unmodified at lysine 4 (Otani et al., 2009;Zhang et al., 2010). Since the ADD domains of DNMT3A/B andDNMT3L clearly show the same binding specificity, the recruit-ment of DNMT3A to chromatin with unmethylated H3 tail isprobably not the primary function of DNMT3L (Zhang et al.,2010).

Jurkowska et al. (2011b) reported that DNMT3A formsoligomers predicted to bind several DNA molecules oriented inparallel and required to tightly tie to heterochromatin by theADD and PWWP domains, which recognize H3 tails unmodi-fied at lysine 4 and H3 trimethylated at lysine 36, respectively(Dhayalan et al., 2010; Zhang et al., 2010). In addition, the associ-ation of the DNMT3A oligomers to DNMT3L changes its subnu-clear localization, from heterochromatin to euchromatin, therebyincreasing its availability and DNA methylation activity forthe generation of DNA methylation imprints (Jurkowska et al.,2011b).

DNMT1Whereas DNMT3 family works as de novo methylases, DNMT1maintains the existing pattern of methylation during chromo-somes replication (Chen and Li, 2006; Jurkowska et al., 2011a)and repair (Mortusewicz et al., 2005; Loughery et al., 2011).Human DNMT1, a large enzyme of 1616 amino acids, isthe most abundant DNMT involved in preserving and prop-agating the existing methylation patterns during cell division(Jurkowska et al., 2011a; Kar et al., 2012). It shows a preferencefor hemimethylated DNA (Goyal et al., 2006) and it is localizedat the DNA replication foci during the S-phase of cell cycle(Leonhardt et al., 1992). In addition, DNMT1, being a highlyprocessive enzyme, is able to methylate long stretches of DNAwithout dissociation (Hermann et al., 2004; Vilkaitis et al., 2005).These two features make it suitable for its role in the maintenanceof methylation patterns. The expression of DNMT1 is ubiquitousand high in proliferating cells and varies in a cell-cycle-dependentmanner, being maximal during the S-phase and extremely lowin non-dividing cells (Robertson et al., 1999; Kimura et al., 2003).Concerning the subcellular localization of DNMT1, it is diffuselydistributed in the nucleus during the interphase and moves tothe replication foci for early and mid S-phase creating a char-acteristic punctuate pattern (O’Keefe et al., 1992; Easwaran et al.,2008). DNMT1 mouse knockout shows extensive demethylationof the genome and embryonic lethality shortly after gastrula-tion, underlying the crucial role of DNMT1 in early development.Embryonic stem cells lacking DNMT1 are viable, despite the lowlevel of DNA methylation, but die after differentiation induc-tion (Li et al., 1992; Chen et al., 1998). Complete inactivation ofDNMT1 in human colorectal carcinoma cells leads to severemitotic defects and progressive cell death (Chen et al., 2007).

Mutations affecting the DNMT1 gene have been associ-ated with two distinct autosomal dominant neurodegenerativediseases: hereditary sensory and autonomic neuropathy withdementia and hearing loss type 1E (HSN1E) and autosomaldominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN;






Klein et al., 2011; Winkelmann et al., 2012). All these mutationsare localized in the DNA replication foci targeting sequence(RFTS) domain, essential inmediating the association of DNMT1to heterochromatin. Mutations in exon 20 of DNMT1 have beenassociated with HSN1E, whereas mutations in exon 21 have beenfound in ADCA-DN (Klein et al., 2013).

From the structural point of view, DNMT1 is a multi-domain enzyme composed by a N-terminal regulatory regionand a C-terminal catalytic domain joined by a series ofKG (lysine-glycine) repeats (Figure 2; Dhe-Paganon et al., 2011;Jurkowska et al., 2011a; Kar et al., 2012). The N-terminal portioncontains several motifs:

• DMAP1 (DNA methyltransferase associated protein 1)charged rich domain is involved in the interaction of DNMT1with the transcriptional repressor DMAP1 as well as in thestability of the enzyme and in its binding to DNA CpG sites(Rountree et al., 2000; Ding and Chaillet, 2002).

• PBD (PCNA-proliferating cell nuclear antigen-binding)domain address DNMT1 to the replication foci through theinteraction between DNMT1 and PCNA.

• NLS (nuclear localization sequence) are at least three.• RFTS (replication foci targeting sequence) is crucial for the

localization of DNMT1 at the centromeric chromatin andreplication foci as well as for its dimerization (Easwaran et al.,2008; Fellinger et al., 2009).

• CXXC zinc domain, similar to the cysteine-rich motif presentin other chromatin associated proteins. It contains eight con-served cysteine residues clustered in two CXXCXXC repeatsthat bind to two zinc ions and is necessary for the recognitionof unmethylated CpG, thus influencing the catalytic activity ofthe enzyme (Pradhan et al., 2008).

• PBHD (polybromo homology domain) domain is composedby the BAH1 and BAH2 (Bromo-adjacent homology 1 and2) motifs, typical of proteins involved in transcriptional reg-ulation. It has been proposed to act as a protein–proteininteraction module specialized in gene silencing even if itsfunctional role in DNMT1 remain unknown.

Thus, the N-terminal regulatory region is designed to rec-ognize the methylation target site (DMAP1, PBD, and RFTSdomains), to bind nucleic acids trough an allosteric site (CXXCdomain) and to allow interaction with other proteins (PBHDdomains), making DNMT1 ideally suited for its role as themaintenance methyltransferase.

The KG linker between the N- and C-terminal regions of theenzyme is composed by a series of lysine and glycine residues,which might contribute to address DNMT1 to the region next tothe replication fork.

Finally, the C-terminal region encloses the catalytic centerof the enzyme bearing the conserved motifs I–X, and is foldedin large and small domains, separated by a huge cleft. Thelarge domain (motifs I-VIII and part of motif X) participates inSAM cofactor binding substrate (cytosine) targeting and otheressential catalytic events. The small domain includes a variableregion between motif VIII and IX, named TRD (target recog-nition domain), the conserved motif IX and part of motif X.

The catalytic domain allows the binding of the target DNA inthe active site and various other regulatory molecules in theallosteric sites, which support a multiple levels of enzyme regula-tion (Dhe-Paganon et al., 2011; Jurkowska et al., 2011a; Kar et al.,2012).

In order to avoid runaway methylation, DNMT1 undergoesto several auto-inhibitory mechanisms. In particular, to keepDNMT1 inactive the RFTS domain is positioned and stabilizeddeep inside the catalytic domain in a task where the hemimethy-lated DNA is expected to fit, thus, masking the catalytic core ofthe enzyme (Qin et al., 2011; Syeda et al., 2011; Bashtrykov et al.,2014). The auto-inhibitory function of the CXXCdomain is morecontroversial. Song et al. (2011). reported that CXXC specificallybinds to unmethylated CpG, activating the CXXC-BH21 linker,which then occupies the catalytic pocket and interferes with itsfunction. By contrast, Bashtrykov et al. (2012) refused this auto-inhibitory mechanism, proposing instead that the recognition ofthe hemimethylated state of target sites resides within the cat-alytic domain. While the unmethylated DNA emerges from thereplication fork, DNMT1 is tethered and kept auto-inhibited toavoid unauthorized de novo methylation of the DNA. As soon asthe hemimethylated sites are released from the replication com-plex, the TRD region of DNMT1 in combination with UHRF1(ubiquitin-like with PHD and ring finger domains 1) recognizesand marks them as prime targets for methylation to guaranteethe correct methylation patterns across successive generations(Kar et al., 2012).

DNMT2DNA (cytosine-5)-methyltransferases 2 is the smallest mam-malian DNMT, now named TRDMT1, which has the poten-tial to methylate RNA instead of DNA (Goll et al., 2006;Schaefer and Lyko, 2010). It completely lacks the regulatory N-terminal region and the C-terminal domain contains all 10sequence motifs that are conserved among DNMTs, includ-ing the consensus S-adenosyl-L-methionine-binding motifs(Okano et al., 1998; Dong et al., 2001; Figure 2). Despite the highsequence and structural similarity with other DNMTs and itscapability to bind the DNA, DNMT2 has failed to show a signifi-cant transmethylase activity (Hermann et al., 2003). Nonetheless,it methylates cytosine 38 in the anticodon loop of tRNAAsp

(Goll et al., 2006), by using a DNA methyl transferase-like cat-alytic mechanism (Jurkowski et al., 2008). Thus, DNMT2 seemsto have intermediate properties between a DNA methyl trans-ferase and a RNA methyl transferase, with which it shares thestructural/catalytic features and the nuclear-cytoplasmic localiza-tion, respectively (Schaefer et al., 2008).

Stochastic Model of DNA MethylationFor long time the classical model of DNA methylation atCpG sites in mammals, with the involvement of both DNMT3and DNMT1 (i.e., de novo and maintenance DNA methyl-transferases, respectively), has been considered a paradigmfor epigenetic information transfer (Holliday and Pugh, 1975;Riggs, 1975). Over the years, several experimental observa-tions highlighted that the site-specific maintenance methyla-tion model had to be revised. Thus, Jeltsch and Jurkowska






(2014) proposed a modified stochastic DNA methylationmodel which includes a new description of both de novoand maintenance methylation process at CpG and non-CpGsites.

An efficient methylation of both DNA strands, starting fromunmethylated DNA, can be achieved by the tight cooperationof DNMT3 enzymes with DNMT1. Thus, de novo and mainte-nance methylation could not be regarded as two distinct events.DNMT3 enzymes, and in particular DNMT3A, can bind adjacentDNA molecules, but only one of the two strands is preferentiallymethylated. By contrast, the hemi-methylated DNA is the favoritesubstrate for DNMT1, which copies the methylation on the sec-ond strand (Fatemi et al., 2002). In addition, DNMT1 showsconsiderable de novomethylation activity on unmethylated DNAboth in vitro (Goyal et al., 2006) and in vivo, as observed inDNMT3A/3B double knockout embryos (Okano et al., 1999).Arand et al. (2012). have confirmed these results by reportingDNMT1-dependent de novo methylation in cells with singleor combined DNMTs knockout. On the other side, deletion ofDNMT3A/3B or DNMT3B alone led to a reduction of main-tenance DNA methylation at repetitive elements, despite thepresence of functional DNMT1 (Chen et al., 2003; Dodge et al.,2005; Arand et al., 2012).

Thus, neither de novo methylation nor maintenance methy-lation can be exclusively assigned to DNMT3A/3B or DNMT1,respectively, but a synchronized cooperation of all the DNMTsseems crucial.

The classical model of maintenance methylation assumes thatthe methylation patterns of single CpG sites are stably inheritedand that exists for this purpose a perfect enzyme, characterized bya working efficiency of 100%.

In reality, average methylation densities of DNA regionsare maintained, instead of exact CpG site-specific methylationpatterns (Zhang et al., 2010), and changes in methylation lev-els occur through stochastic processes (Landan et al., 2012). Infact, despite the 10–40-fold preference of DNMT1 for hemi-methylated DNA, this feature is not enough to copy accuratelythe site-specific methylation status of around 56million CpG sitesin the human genome over several rounds of DNA replication(Jeltsch and Jurkowska, 2014).

In addition, patterns of non-CpG DNA methylation, typicalof plants, have been reported also in the human genome, takingplace in CpA sites, and specifically introduced by DNMT3A. Thisclearly indicates a permanent de novomethylation activity of thisenzyme, which is not in agreement with the classical methylationmodel (Arand et al., 2012; Guo et al., 2014).

Finally, also the DNA demethylation has to be considered inthe establishment and maintenance of DNAmethylation pattern.This process could be passive, during the replication, or active,performed by the family of ten eleven translocation (TET) dioxy-genases. These enzymes oxidize the methyl groups of methyl-cytosine and are expressed both during early development andalso in later stages, suggesting a permanent DNA demethyla-tion. Hydroxymethylation of DNA by TET enzymes has beenproposed to keep CpG islands in unmethylated state by coun-teracting stochastic DNA methylation (Kohli and Zhang, 2013;Wu and Zhang, 2014).

In conclusion, the DNA methylation is influenced by all theevents above described, that are combined in the stochastic modelof Jeltsch and Jurkowska (2014):

• DNA methylation at each site is determined by the local ratesof methylation and demethylation

• The local rates of methylation depend on targeting/regulationof DNMTs/demethylases and on chromatine remodel-ing/DNA accessibility

• DNA methylation occurs on both CpG and non-CpG sites denovo and maintenance methylation are combined in a unifiedmechanism

• The average DNA methylation level of DNA regions is inher-ited rather than the methylation state of individual CpGsites.

Mitochondrial DNA Methylation

Unlike nuclear DNA, the methylation of mtDNA is a highlycontroversial topic that after 40 years is still matter of debateamong researchers. In fact, although recent studies have demon-strated the presence of 5 mC in mtDNA, skepticism regardingthe sensitivity and reproducibility of the methods used and theputative biological function of methylation in mitochondria stillremains. Indeed, the observed methylation levels are very low(1–5%) and, furthermore, mtDNA has a different organizationcompared to nuclear DNA (i.e., no histones, lack of introns,multicopy genomes), which should imply a different regula-tory mechanism of gene expression through DNA methylation.In fact, mammalian cells typically contain 1,000–10,000 copiesof mtDNA, which are organized into nucleoprotein complexestermed nucleoids, being TFAM (mitochondrial transcriptor fac-tor A) the main protein component. The TFAM/mtDNA ratiofinely regulates the fraction of active mtDNA molecules availablefor mitochondrial replication/transcription (Bogenhagen, 2012;Campbell et al., 2012; Farge et al., 2014).

The first approaches to investigate mtDNA methylation dateback to 1970s, when Vanyushin et al. (1971) found a DNMTactivity in mitochondria isolated from loach embryos, demon-strated the presence of 5 mC in mtDNA extracted from beef heart(Vanyushin and Kirnos, 1974) and evidenced a different speci-ficity for DNA methylases isolated from nucleus and mitochon-dria, being the mitochondrial specific for mono-pyrimidines andthe nuclear for di- and tri-pyrimidines (Vanyushin and Kirnos,1977). However, in the same years the absence of 5 mC inmtDNA from mouse, hamster, frog, and HeLa cells was alsoreported (Nass, 1973; Dawid, 1974). Shmookler and Goldstein(1983) and Pollack et al. (1984), studying both human fibroblastsand mouse fibroblastoid cells, clarified that methylation occurredin mtDNA with a frequency of 1.5–5% and only in CpG di-nucleotides, which, however, are underrepresented in mtDNA(Pollack et al., 1984; Cardon et al., 1994). After many years, in2004, the presence of methylated cytosines in mtDNA, from gas-tric and colorectal cancers, was once again denied using bisulfite-PCR-single-stranded DNA conformation polymorphism onthree selected regions of mtDNA containing 37 CpG sites(Maekawa et al., 2004).






Rebelo et al. (2009) failed to observe mtDNA methylation inhuman osteosarcoma 143B, HEK293, and HeLa cells, under stan-dard culture conditions, but the same authors also reasonedthat percentages of methylation <5% have not been detectedby the approach employed (site-specific methylation restric-tion enzymes). However, in this study, some methylation ofmtDNA was observed after the induced-expression of two dif-ferent mitochondria-targeted bacterial methyltransferases and,additionally, the levels of this methylation increased duringmtDNA replication, when it is assumed that nucleoids are remod-eled and mtDNA could be less protected by proteins (i.e., TFAM)and more accessible to DNMTs (Rebelo et al., 2009).

The issue of mtDNA methylation came back again in2011, when two different groups demonstrated the presenceof 5 mC and DNMTs in mitochondria through novel andmore sensitive approaches (Chestnut et al., 2011; Shock et al.,2011). Surprisingly, a mitochondria targeted DNMT1 isoform(mtDNMT1), conserved in different species, has been foundby Shock et al. (2011); the mtDNMT1 seems to be translatedfrom an unconventional ATG site, located immediately upstreamof the canonical one, and localized to mitochondria where itbounds to mtDNA. Moreover, the mtDNMT1 gene expressionwas induced by NRF1 and PGC1α, two master regulators ofmitochondrial biogenesis, in HCT116 cells (Shock et al., 2011).Interestingly, gene expression of mtDNA-encoded genes wasaltered after mtDNMT1 over-expression, with reduced levels ofMT-ND6 in the L-strand and increased levels of MT-ND1in theH-strand, suggesting an opposite role for mtDNMT1, and cyto-sine methylation in the two strands; however, the expressionlevels of MT-CO1 and MT-ATP6, both in the H-strand, wereunaltered (Shock et al., 2011). The authors speculate that mtD-NMT1 interferes with MTERF-dependent transcription termina-tion, inducing an increased transcription of MT-ND1 throughthe HSP1, with no effects on the polycistronic mRNA producedby HSP2. Lastly, in this study the presence of 5 mC in mtDNAwas demonstrated by a methylated DNA-immunoprecipitation(MeDIP) approach, followed by real time-PCR (Shock et al.,2011).

Concomitantly, Chestnut et al. (2011) demonstrated the pres-ence of DNMT3A in mitochondria isolated from mouse brainand from human motor cortex, whereas DNMT1 was faintlydetectable in mitochondria from these tissues. Furthermore,co-localization of 5 mC and mitochondria was shown byimmunofluorescence experiments using antibodies recognizing5 mC and a specific mitochondrial marker (superoxide dismutaseisoform 2, SOD2; Chestnut et al., 2011).

The mtDNA methylation was also investigated by a liq-uid chromatography-electrospray ionization tandem-mass spec-trometry (LC-ESI-MS) method, proving the existence of 5 mC inthe mitochondrial genome (Infantino et al., 2011), and soon afteralso 5 hmC have been identified inmtDNA by an ELISA approach(Dzitoyeva et al., 2012).

In a study focused on the intragenic methylation of PolgA indifferent mouse cells/tissues as possibly correlated with PolgAexpression and mtDNA content regulation, the authors foundin the eluted fraction of MeDIP analysis some mtDNA encodedgenes (mt-cytb, mt-co1) and the D-loop region, indicating that

these genes have 5 mC and 5 hmC, although at very low levels(Kelly et al., 2012).

Pirola et al. (2013) further evidence of methylated mtDNA hasbeen published. The methylation of MT-ND6, MT-CO1 and ofthe D-loop of mtDNA was assessed by quantitative methylationspecific-PCR in the context of non-alcoholic fatty liver disease.The authors found a significant association between the conditionof non-alcoholic steatohepatitis (NASH) and the methylation ofMT-ND6 gene, which inversely correlates with MT-ND6 tran-scription and protein expression in the liver of subject affectedby NASH (Pirola et al., 2013).

The methylation of the D-loop region was also confirmedin mammals by bisulfite-sequencing (30 clones for each sam-ple sequenced) and by MeDIP (Bellizzi et al., 2013). Methylationwas limited to the L-strand of the D-loop and the majority ofmethylated cytosines were located outside the CpG nucleotides;different tissues were analyzed (blood cells, fibroblasts, HeLacells) and tissue-specific patterns of methylation were identi-fied, HeLa cells showing the highest percentage of methylatedcytosines (Bellizzi et al., 2013). In the same study, DNMT1 andDNMT3B expression in mitochondria from HeLa and mouse3T3-L1 cells was evidenced (Bellizzi et al., 2013).

On the contrary, Wong et al. (2013) proved that in mouseskeletal muscle DNMT1 was not present within the organelle,but probably bound to the outer membrane (detectable in thecrude mitochondrial fraction), whereas DNMT3A was present inthe pure mitochondrial fraction isolated from both mouse skele-tal muscle and spinal cord; DNMT3B was not detectable in anyof the mitochondrial preparations. DNMT3A was also presentin mitochondria from human cerebral cortex, but not in mito-chondria from HEK293 cells, showing a preferential expressionin mitochondria from excitable tissues in humans and mouse(Wong et al., 2013). In addition, methylated cytosines in themouse D-loop and mt-rnr2 (coding for 16S RNA) were iden-tified by bisulfite treatment and pyrosequencing, and differentlevels of methylation in brain (highest percentage of 5 mC),liver and testes were shown (Wong et al., 2013). Moreover, aspreviously observed by Chestnut, the co-localization of 5 mC,and mitochondria by immunofluorescence was confirmed andinterestingly, it was observed a co-localization of 5 mC withautophagosome (LC3Apositive staining), indicating thatmtDNAmethylation may be involved in mitophagy (Wong et al., 2013).

Also Byun et al. (2013) carried out bisulfite-pyrosequencing toinvestigate methylation of three mtDNA regions, MT-TF (tRNAphenylalanine gene, two CpG sites), MT-RNR1 (12S RNA gene,two CpG sites), and the D-loop (three CpG sites), in associationwith effect of airborne pollutants. The authors were able to detectaround 5–6% of 5 mC in the selected regions.

Hong et al. (2013) critically revised all methods employedto quantify methylated mtDNA in the last years. Contextually,these authors established the absence of CpG methylation inhuman mtDNA performing bisulfite-sequencing and analyzingdata of bisulfite-next generation sequencing (NGS) previouslypublished by Akalin et al. (2012) andHong et al. (2013). Bisulfite-sequencing failed to reveal 5 mC in four selected mtDNA regions(MT-RNR1, MT-RNR2, MT-CO2, MT-ATP6) of HEK293 cells,and identified very low frequencies of 5 mC in the same regions






of HCT116 and blood cells mtDNA (<0.5%), considered notrelevant because comparable to the non-convertion rate of bisul-fite; cross-contamination of nuclear DNA sequences of mito-chondrial origins (NUMTs; Lascaro et al., 2008) has been alsoexcluded based on the specificity of the analyzed regions formtDNA sequence (Hong et al., 2013). Comparable levels of 5 mC(<0.5%), both in CpG or not-CpG sites, came from the re-analysis of bisulfite-NGS data on HCT116 cells previously pub-lished (Akalin et al., 2012), but the mean coverage obtained of94x may be not sufficient, considering that HCT116 cells havea mtDNA content of about 4000 copies, as mentioned by theauthors (Hong et al., 2013).

Lastly, a comparative analysis of mitochondrial methylomesin 39 different cell lines was carried out extracting publicdata from the NIH Roadmap Epigenomics project (Ghosh et al.,2014). The re-analyzed data, deriving from MeDIP-sequencingincluded human brain, breast, blood, penis, and two cell lines,H1 and neurosphere cultured cells; moreover, some tissueswere analyzed at different developmental time points. Theauthors identified tissue- and development-specific pattern of5 mC methylation in mtDNA, with MT-ND6 and MT-ATP6showing progressive reduction in methylation correlated withbrain development. However, also in this case, methylatedcytosines resulted underrepresented (<0.5%) and an extremely

low minimum coverage of 5x was considered (Ghosh et al.,2014).

In conclusion, since 2011 several studies claimed the occur-rence of mtDNA methylation, but a few others also assertedthe absence of 5 mC in mtDNA (Figure 3). Many of the pub-lished approaches had some limitations, such as the reproducibil-ity of the methods based on the use of antibodies (ELISA,MeDIP), or the enrichment by amplification before sequenc-ing that may create a bias, or the very low coverage con-sidered for the analysis of NGS data that may influence thequantification of 5 mC referred to total mtDNA copies. Infact, the analysis of mtDNA methylation should take intoaccount that the mitochondrial genome is multicopy, differ-ing for copy number depending on cell types and tissues(Campbell et al., 2012). None of the sequencing studies citedabove have considered the percentage of methylated sites respectto total mtDNA molecules present in the cells/tissues analyzed(“methylation heteroplasmy”), but only the percentage of 5 mCrespect to total cytosines, as is usually done for the nucleargenome.

The scenario becomes even blurrier when considering thethe controversial results on DNMTs localization (Tables 1 and 2),since DNMT1 presence within mitochondria was demonstrated(Shock et al., 2011; Bellizzi et al., 2013), and then denied by

FIGURE 3 | Chronological sequence of mtDNA methylation studies. Schematic representation of all the studies which support or denied the mtDNAmethylation.






TABLE 1 | Mitochondrial localization of DNA(cytosine-5)-methyltransferases – DNMT1, DNMT3A, and DNMT3B, asevaluated by Western blot, in different human cell types and tissues.

DNA methyl-transferase

Mitochondriallocalization

Organism Celltype/tissue

Reference

DNMT1 Yes Human HCT116 Shock et al.(2011)

DNMT1 Yes Human HEK293 Chestnut et al.(2011)

DNMT1 Yes Human HeLa Bellizzi et al.(2013)

DNMT3A Yes Human HEK293 Chestnut et al.(2011)

DNMT3A Yes Human Frontalcortex

Wong et al.(2013)

DNMT3A No Human HEK293 Wong et al.(2013)

DNMT3B Yes Human HeLa Bellizzi et al.(2013)

TABLE 2 | Mitochondrial localization of DNMT1, DNMT3A, and DNMT3B,as evaluated by Western blot, in different mouse cell types and tissues.

DNAmethyl-transferase

Mitochondriallocaliza-tion

Organism Cell type/tissue Reference

DNMT1 Yes Mouse MEF Shock et al.(2011)

DNMT1 Yes Mouse NSC34 AstrocyteMicroglia

Chestnut et al.(2011)

DNMT1 Yes Mouse 3T3-L1 Bellizzi et al.(2013)

DNMT1 No Mouse Adult skeletalmuscle

Wong et al.(2013)

DNMT3A No Mouse MEF Shock et al.(2011)

DNMT3A Yes Mouse NSC34 AstrocyteMicroglia

Chestnut et al.(2011)

DNMT3A Yes Mouse Adult skeletalmuscle, brain,spinal cord, heart,testes, spleen

Wong et al.(2013)

DNMT3B No Mouse MEF Shock et al.(2011)

DNMT3B Yes Mouse 3T3-L1 Bellizzi et al.(2013)

DNMT3B No Mouse Adult skeletalmuscle

Wong et al.(2013)

Wong et al. (2013), who in addition observed DNMT3A withinmitochondria, but not DNMT3B, which, however, has beendetected in these organelles by others (Bellizzi et al., 2013).Therefore, it remains truly unclear which DNMTs might beimplicated in mtDNAmethylation. Overall, further experiments,focused on both global/site-specific methylation and the func-tional effects of this modification are necessary to unequivo-cally demonstrate methylation of the mitochondrial genome,its biological function, and the possible links to pathologicalconditions.

Defective DNA Methylation andNeurological Diseases

Altered DNA methylation is a common hallmark of cancer, and200, 590, and 320 somatic mutations in DNMT1, DNMT3A,and DNMT3B, respectively, have been reported in the catalogof somatic mutations in cancers (COSMIC). Up and down-regulation of DNMTs has also been observed in different types ofcancer (Forbes et al., 2008; Subramaniam et al., 2014; Wu et al.,2014).

In addition to cancer, alterations of the DNA methylationmachinery also cause a few neurodegenerative and neurodevel-opmental diseases, some of these recently described thanks towhole-exome sequencing (Klein et al., 2011; Winkelmann et al.,2012; Tatton-Brown et al., 2014). Genetic defects may producean epigenetic deregulation at different levels, affecting boththe enzymes responsible for de novo/maintenance methylation(DNMT3/DNMT1), or proteins with a role in the recogni-tion and binding of CpG methylated sites (MBDs, MeCP2;Weissman et al., 2014). As mentioned previously, in Klein et al.(2011) and Winkelmann et al. (2012), two independent exome-sequencing studies revealed mutations in the RFTS domain ofDNMT1 gene causing two neurodegenerative disorders withoverlapping features: hereditary sensory HSN1E and ADCA-DN.More recently, de novo mutations in DNMT3A gene affectingfunctional conserved domains of the protein, have been iden-tified by exome-sequencing in patients with overgrowth dis-orders (Tatton-Brown et al., 2014), resembling clinical featuresof histone defects and imprinting disorders (Weissman et al.,2014).

Besides primary epigenetic defects, deregulation of DNAmethylation may also influence the pathogenesis of other neu-rodegenerative disorders, like amyotrophic lateral sclerosis (ALS)or Alzheimer disease (AD), Parkinson disease (PD), or neurode-velopmental diseases, such as Down syndrome (DS; Lu et al.,2013). Global DNA hypomethylation in AD has been evi-denced in a post-mortem study of monozygotic twins discordantfor AD (Mastroeni et al., 2009) and later confirmed by others(Sung et al., 2011; Chouliaras et al., 2013), whereas global hyper-methylation has been described in other cases (Bakulski et al.,2012; Rao et al., 2012; Coppieters et al., 2014). In contradic-tion, Lashley et al. (2014) excluded alterations in global DNAmethylation and hydroxymethylation in AD. Hypomethylationof specific genes related to AD (i.e., PSEN1, NEP, BIN1) havealso been observed (Lu et al., 2013; Yu et al., 2014). An addi-tional link between DNA methylation and AD came from theassociation of the chromosome location of DNMT1 (19p13.2)with familial late-onset AD (FLOAD; Wijsman et al., 2004),although sequencing of exon 20 and 21 of DNMT1 in 364FLOAD cases failed to identify pathogenic mutations in theseregions, but only known polymorphisms with expected minorallele frequencies based on European HapMap data (Klein et al.,2013).

Hypomethylation of intron 1 of the α-synuclein (SNCA)gene might be relevant for PD pathogenesis, inducing anincreased expression of α-synuclein in the substantia nigraof PD patients and possibly contributing to the Lewy Body






formation (Jowaed et al., 2010; Matsumoto et al., 2010; Lu et al.,2013). Moreover, low levels of nuclear DNMT1 in post-mortembrain of PD patients have been observed, indicating a pos-sible link between SNCA hypomethylation and the methylase(Desplats et al., 2011).

DNA methylation seems to have a role also in the pathogen-esis of ALS, as proposed by Chestnut et al. (2011), by demon-strating that the levels of DNMT3A, DNMT1, and 5 mCare increased in motor neurons of ALS affected patients.Furthermore, abnormal mtDNA methylation has been found inALS (Wong et al., 2013) and also in patients affected by Down’ssyndrome (Infantino et al., 2011), and for both diseases a mito-chondrial dysfunction has been documented (Valenti et al., 2011;Cozzolino et al., 2013).

Based on the mounting evidence of mitochondrial dysfunc-tion in AD and PD, including organelle bioenergetics, dynamics,and quality control (Schon and Przedborski, 2011; Burté et al.,2015), DNAmethylation may influence the pathogenesis of theseneurodegenerative diseases, acting both on nuclear and mito-chondrial DNAs. Moreover, mitochondria participate in the pro-duction of the universal methyl donor SAM, through synthesisof ATP and folate, whose deficiency, together with high levelsof homocysteine, have been associated with dementia, and neu-rodegenerative diseases, including AD and PD (Iacobazzi et al.,2013; Ansari et al., 2014). To the best of our knowledge, mtDNAmethylation has never been investigated in AD and PD, and nei-ther methylation of nuclear genes encoding for mitochondrialproteins.

ADCA-DN and HSN1E: DefectiveMethylation Diseases withMitochondrial Involvement

Hereditary sensory neuropathy with dementia and hearing loss(OMIM 614116), an adult-onset neurodegenerative disorder,is the first Mendelian inherited “methylopathy” identified dueto mutations in the DNMT1 gene affecting the RFTS domain(Klein et al., 2011). Shortly after, another adult-onset neurode-generative disease, ADCA-DN (OMIM 604121), has been asso-ciated to mutations in DNMT1 located in the same func-tional domain (Winkelmann et al., 2012). HSN1E and ADCA-DN have been initially considered as two distinct clinicalentities, but more recently the evidence of overlapping clini-cal features, often subclinical, has emerged, strongly suggest-ing that they can be better considered as phenotypes belong-ing to the same neurodegenerative spectrum (Moghadam et al.,2014).

Hereditary sensory neuropathy with dementia and hearingloss is a severe disorder characterized by both central and periph-eral nervous system involvement, with peripheral neuropathyleading to extremity injuries and infections, frequently need-ing amputations, severe early onset hearing loss, and middle-agedementia (Hojo et al., 1999; Klein et al., 2011). By a whole-exomesequencing analysis, two heterozygous mutations in DNMT1have been identified as the genetic cause of HSN1E in four unre-lated families from different geographical area (Klein et al., 2011).

A point mutation leading to the p.Tyr511Cys (NP_001124295.1)aminoacid substitution was found in three families, whereasthree nucleotide changes causing the substitution of two con-tiguous aminoacids, p.Asp506Glu-Pro507Arg (NP_001124295.1)were found in the fourth pedigree of this study; both mutationswere located in exon 20 and affected the RFTS domain of DNMT1(Klein et al., 2011). Through an accurate functional investiga-tion, Klein et al. (2011) demonstrated that these mutations causea premature degradation of the protein, reduced methyltrans-ferase activity, and impaired binding to heterochromatin in G2phase, leading to global DNA hypomethylation and site-specifichypermethylation.

The abnormal status of DNA methylationinduced by thep.Tyr511Cys mutation has been thoroughly analyzed by whole-genome bisulfite sequencing in three pairs of HSN1E patientscompared to gender and age-matched siblings, providing anevaluation of methylation at single base-resolution (Sun et al.,2014). The results showed prevalent hypomethylation in inter-genic regions and around the transcription start sites, with thehighest reduction of methylation in chromosomes X and 18(Sun et al., 2014). Furthermore, using the Ingenuity PathwayAnalysis, the differentially methylated regions identified werelinked to “neurological disease,” including progressive neu-ropathy, PD, AD, ALS, narcolepsy (NC), “psychological disor-ders,” “skeletal and muscular disorders” and “cancer,” whereasthe most compromised pathway resulted the NAD+/NADHmetabolism, which is also implicated in neurodegeneration(Sun et al., 2014).

In 2013 two additional cases of HSN1E with psychiatricmanifestations and seizures were identified, again caused bymutations in the exon 20 of DNMT1, one of them previ-ously identified (p.Tyr511Cys), and one previously unreportedmutation p.Tyr511His (NP_001124295.1), affecting both thesame amino acid residue in the RFTS domain (Klein et al.,2013). Two other HSN1E families has been described in2014, presenting mutations in exon 20 of DNMT1: onepoint mutation (p.Pro506Arg, NP_001124295.1) hitting thesame amino acid position of another previously reportedcase (Klein et al., 2011), and a novel trinucleotide deletion(p.Lys521del, NP_001124295.1; Moghadam et al., 2014). Theextensive characterization of the HSN1E patients in this latterstudy showed that subclinical symptoms usually characterizingADCA-DN, such as NC (without cataplexy and with normalhypocretin-1 level in cerebrospinal fluid) and optic atrophy, maybe present in patients affected by HSN1E, highlighting aphe-notypic overlap between these two diseases (Moghadam et al.,2014).

Moreover, the strict association between exon 20 and HSN1Ehas been broken by Yuan et al. (2013) a unique case of HSN1Ecaused by a novel mutation (p.His569Arg, NP_001124295.1) inexon 21, that is usually associated with ADCA-DN), but stillaffecting the RFTS domain and further remarking the idea of acontinuum.

Autosomal dominant cerebellar ataxia-deafness andnarcolepsy was first described by Melberg et al. (1995),but the genetic cause has been identified only recently inWinkelmann et al. (2012). ADCA-DN is initially characterized






by late-onset NC with or without cataplexy, complicated in laterstages of the disease by sensorineural deafness, cerebellar ataxia,and dementia appear (Melberg et al., 1995). Mild polyneu-ropathy, optic atrophy, epilepsy, psychosis, diabetes mellitus,cardiomyopathy, and progressive cerebral, cerebellar, andbrainstem atrophy may also be present (Melberg et al., 1999;Winkelmann et al., 2012; Moghadam et al., 2014). DNMT1mutations in exon 21 have been found to cause ADCA-DN(Winkelmann et al., 2012), thus qualifying DNMT1 as the thirdmutant leading to genetically determined NC (Peyron et al.,2000; Hor et al., 2011). A previous link between DNMT1and NC emerged from a genome-wide study associatingNC with the SNP rs4804122, located in a region of highlinkage disequilibrium spanning several genes includingDNMT1, and a weak correlation between this NC-associatedallele and lower DNMT1 mRNA expression was also docu-mented in peripheral blood mononuclear cells (Kornum et al.,2011).

Initially, three missense mutations have been identified in fourdifferent families, all affecting the RFTS domain: p.Ala570Val,p.Val606Phe, p.Gly605Ala (NP_001124295.1). The authors spec-ulated that these mutations might affect the interaction withother proteins, i.e., HDAC2, or the DNA- binding, beingclose to three phenylalanines critical for the anchoring ofthe RFTS domain to the DNA-binding pocket (Takeshita et al.,2011; Winkelmann et al., 2012). Concerning the role of mutantDNMT1 in the pathogenesis of NC in ADCA-DN, hypocre-tin cells, whose loss cause NC, may be particularly suscepti-ble to altered methylation induced by the DNMT1 mutations(Winkelmann et al., 2012). Alternatively, the mutations mayimpair the regulation and differentiation of immune cells pos-sibly promoting an autoimmune reaction (Winkelmann et al.,2012), thus supporting autoimmunity as the main pathogenicmechanism of NC (Fontana et al., 2010). This hypothesis isreinforced by the critical role played by DNMT1for T-cellsdevelopment, function and survival (Lee et al., 2001; Wang et al.,2013). However, the HLA-DQB1∗06:02, which represents themajor genetic risk factor for NC (Tafti et al., 2014), was neg-ative in all except for two of the ADCA-DN patients withDNMT1 mutations (Winkelmann et al., 2012; Pedroso et al.,2013; Moghadam et al., 2014), weakening the autoimmunehypothesis for this disease. An additional case of ADCA-DNdue to a novel missense mutation in exon 21 of DNMT1,p.Cys596Arg (NP_001124295.1), has been reported; this patientdisplayed the typical, previously described features of the disease(Pedroso et al., 2013).

Interestingly, HSN1E and ADCA-DN present common fea-tures resembling mitochondrial encephalomyopathies, suchas sensorineural deafness, optic atrophy, cerebellar involve-ment, and peripheral neuropathy (Moghadam et al., 2014).Furthermore, a mitochondrial dysfunction at the biochemicallevel was already documented in skeletal muscle of an ADCA-DN patient in Melberg et al. (1995). In addition, the alteration ofNAD+/NADH-related pathways that emerged from the methy-lome study on HSN1E (Sun et al., 2014) also reinforces the possi-bility of mitochondrial dysfunction in the complex pathogenesisof this disorder.

Furthermore, hypomethylation of nuclear genes withmitochondrial function has been observed in HSN1E patientscompared to sex and age-matched controls, although a statisticalsignificance was not reached (Sun et al., 2014).

In conclusion, 10 different mutations in exons 20 and 21of the DNMT1 gene have been identified to date, six caus-ing HSN1E and four causing ADCA-DN, two distinct dis-eases now considered as clinical phenotypes of the same dis-ease spectrum (Moghadam et al., 2014). In fact, all the muta-tions affect the RFTS domain, which has a regulatory func-tion for the DNMT1 activity. In particular, mounting evi-dence demonstrate that this domain has an auto-inhibitoryrole (Bashtrykov et al., 2014) and it has been suggested thatits deletion activates DNMT1 for euchromatic DNA-binding,but at the same time decreases heterochromatin binding prob-ably through a missing protein interaction, thus producingpassive DNA demethylation (Wu et al., 2014). Moreover, theRFTS deletion seems to produce a methylation status typicalof cancer, with global hypomethylation and promoter hyper-methylation, and additional 26 mutations in this domain havebeen found in different tumors (Forbes et al., 2008; Wu et al.,2014).

Open questions still have to be addressed. For example, itis unclear whether mutations in exon 20 and 21 of DNMT1act through the same pathogenic mechanism, and the methyla-tion status of ADCA-DN patients has not been investigated yet.Furthermore, although the altered methylation pattern observedin HSN1E patients resembles that of tumorigenesis (Sun et al.,2014) and loss of function of RFTS domain enhances tumori-genicity (Wu et al., 2014), none of the DNMT1 mutant patientsseem to develop cancer. Lastly, the possible role of mitochon-drial dysfunction deserves to be unraveled by functional studies,to shed light on the potential role of mito-epigenetics (fromboth nuclear and mitochondrial genomes) in the pathogenicmechanisms of DNMT1mutations. Tackling all these open ques-tions will be crucial to understand the pathogenesis of DNMT1related neurodegeneration, including the narcoleptic features,but ultimately will also help to resolve the question of mtDNAmethylation.

Final Remarks

The fascinating field of epigenetic regulation of gene expressionis fast evolving, as well as the implications in human pathol-ogy. One point highlighted by reviewing the state of art of thisfield, and prompted by the recent identification of neurodegen-erative disorders due to pathogenic mutations in the DNMT1gene, is the role played by the mitochondrial methylome as awhole, includes obviously the tissue and cell-specific methylationpattern of the nuclear mitochondrial proteome (about 1500–2000 nuclear genes), and the still very controversial existenceof mtDNA methylation. This latter longstanding question needsto be resolved, and the possible biological function played bymtDNA methylation in cell physiology may add to the com-plex inter-genomic dialog between nuclear and mitochondrialDNAs. The rapidly evolving NGS approaches should allow the






resolution of the controversies, in conjunction with func-tional studies, paying attention to the peculiar features of themitochondrial genome, which include gene organization, tran-scription, and replication, and most importantly its multicopynature. The nuclear counterpart represented by the growing

list of nuclear genes implicated in mitochondrial biology isequally crucial for understanding the complexities of can-cer and neurodegeneration, now fuelled by the existence ofan ideal model to investigate, the DNMT1-related humandiseases.

References

Akalin, A., Garrett-Bakelman, F. E., Kormaksson, M., Busuttil, J., Zhang, L.,Khrebtukova, I., et al. (2012). Base-pair resolution DNA methylation sequenc-ing reveals profoundly divergent epigenetic landscapes in acute myeloidleukemia. PLoS Genet. 8:e1002781. doi: 10.1371/journal.pgen.1002781

Ansari, R., Mahta, A., Mallack, E., and Luo, J. J. (2014). Hyperhomocysteinemiaand neurologic disorders: a review. J. Clin. Neurol. 10, 281–288. doi:10.3988/jcn.2014.10.4.281

Arand, J., Spieler, D., Karius, T., Branco, M. R., Meilinger, D., Meissner, A.,et al. (2012). In vivo control of CpG and non-CpG DNA methylation byDNA methyltransferases. PLoS Genet. 8:e1002750. doi: 10.1371/journal.pgen.1002750

Bakulski, K. M., Dolinoy, D. C., Sartor, M. A., Paulson, H. L., Konen, J. R.,Lieberman, A. P., et al. (2012). Genome-wide DNA methylation differencesbetween late-onset Alzheimer’s disease and cognitively normal controls inhuman frontal cortex. J. Alzheimers Dis. 29, 571–588. doi: 10.3233/JAD-2012-111223

Bashtrykov, P., Jankevicius, G., Smarandache, A., Jurkowska, R. Z., Ragozin, S.,and Jeltsch, A. (2012). Specificity of Dnmt1 for methylation of hemimethy-lated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–578. doi:10.1016/j.chembiol.2012.03.010

Bashtrykov, P., Rajavelu, A., Hackner, B., Ragozin, S., Carell, T., and Jeltsch, A.(2014). Targeted mutagenesis results in an activation of DNAmethyltransferase1 and confirms an autoinhibitory role of its RFTS domain. Chembiochem 15,743–748. doi: 10.1002/cbic.201300740

Bellizzi, D., D’Aquila, P., Scafone, T., Giordano, M., Riso, V., Riccio, A.,et al. (2013). The control region of mitochondrial DNA Shows an unusualCpG and non-CpG methylation pattern. DNA Res. 20, 537–547. doi:10.1093/dnares/dst029

Bestor, T. H. (2000). The DNA methyltransferases of mammals. Hum. Mol. Genet.9, 2395–2402. doi: 10.1093/hmg/9.16.2395

Bogenhagen, D. F. (2012). Mitochondrial DNA nucleoid structure.Biochim. Biophys. Acta 1819, 914–920. doi: 10.1016/j.bbagrm.2011.11.005

Burté, F., Carelli, V., Chinnery, P. F., and Yu-Wai-Man, P. (2015). Disturbed mito-chondrial dynamics and neurodegenerative disorders. Nat. Rev. Neurol. 11,11–24. doi: 10.1038/nrneurol.2014.228

Byun, H. M., Panni, T., Motta, V., Hou, L., Nordio, F., Apostoli, P., et al. (2013).Effects of airborne pollutants on mitochondrial DNA methylation. Part FibreToxicol. 10, 18. doi: 10.1186/1743-8977-10-18

Campbell, C. T., Kolesar, J. E., and Kaufman, B. A. (2012). Mitochondrial tran-scription factor A regulates mitochondrial transcription initiation, DNA pack-aging, and genome copy number. Biochim. Biophys. Acta 1819, 921–929. doi:10.1016/j.bbagrm.2012.03.002

Cardon, L. R., Burge, C., Clayton, D. A., and Karlin, S. (1994). Pervasive CpG sup-pression in animal mitochondrial genomes. Proc. Natl. Acad. Sci. U.S.A. 91,3799–3803. doi: 10.1073/pnas.91.9.3799

Cedar, H., and Bergman, Y. (2011). Epigenetics of haematopoietic cell develop-ment. Nat. Rev. Immunol. 11, 478–488. doi: 10.1038/nri2991

Chatterjee, R., and Vinson, C. (2012). CpG methylation recruits sequencespecific transcription factors essential for tissue specific gene expres-sion. Biochim. Biophys. Acta 1819, 763–770. doi: 10.1016/j.bbagrm.2012.02.014

Chen, R. Z., Pettersson, U., Beard, C., Jackson-Grusby, L., and Jaenisch, R. (1998).DNA hypomethylation leads to elevatedmutation rates.Nature 395, 89–93. doi:10.1038/25779

Chen, T., Hevi, S., Gay, F., Tsujimoto, N., He, T., Zhang, B., et al. (2007). Completeinactivation of DNMT1 leads to mitotic catastrophe in human cancer cells.Nat.Genet. 39, 391–396. doi: 10.1038/ng1982

Chen, T., and Li, E. (2006). Establishment and maintenance of DNA methyla-tion patterns in mammals. Curr. Top Microbiol. Immunol. 301, 179–201. doi:10.1007/3-540-31390-7_6

Chen, T., Tsujimoto, N., and Li, E. (2004). The PWWP domain of Dnmt3aand Dnmt3b is required for directing DNA methylation to the major satel-lite repeats at pericentric heterochromatin. Mol. Cell. Biol. 24, 9048–9058. doi:10.1128/MCB.24.20.9048-9058.2004

Chen, T., Ueda, Y., Dodge, J. E., Wang, Z., and Li, E. (2003). Establishmentand maintenance of genomic methylation patterns in mouse embryonicstem cells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605. doi:10.1128/MCB.23.16.5594-5605.2003

Cheng, X. (1995). Structure and function of DNA methyltrans-ferases. Annu. Rev. Biophys. Biomol. Struct. 24, 293–318. doi:10.1146/annurev.bb.24.060195.001453

Cheng, X., and Blumenthal, R. M. (2008). Mammalian DNA methyltransferases: astructural perspective. Structure 16, 341–50. doi: 10.1016/j.str.2008.01.004

Chestnut, B. A., Chang, Q., Price, A., Lesuisse, C., Wong, M., and Martin, L. J.(2011). Epigenetic regulation of motor neuron cell death through DNA methy-lation. J. Neurosci. 31, 16619–16636. doi: 10.1523/JNEUROSCI.1639-11.2011

Chouliaras, L., Mastroeni, D., Delvaux, E., Grover, A., Kenis, G., Hof, P. R., et al.(2013). Consistent decrease in global DNA methylation and hydroxymethyla-tion in the hippocampus of Alzheimer’s disease patients. Neurobiol. Aging 34,2091–2099. doi: 10.1016/j.neurobiolaging.2013.02.021

Coppieters, N., Dieriks, B. V., Lill, C., Faull, R. L., Curtis, M. A., and Dragunow,M. (2014). Global changes in DNA methylation and hydroxymethylationin Alzheimer’s disease human brain. Neurobiol. Aging 35, 1334–1344. doi:10.1016/j.neurobiolaging.2013.11.031

Cozzolino, M., Ferri, A., Valle, C., and Carrì, M. T. (2013). Mitochondria and ALS:implications from novel genes and pathways.Mol. Cell. Neurosci. 55, 44–49. doi:10.1016/j.mcn.2012.06.001

Dawid, I. B. (1974). 5-methylcytidylic acid: absence from mitochondrial DNA offrogs and HeLa cells. Science 184, 80–81. doi: 10.1126/science.184.4132.80

Denis, H., Ndlovu, M. N., and Fuks, F. (2011). Regulation of mammalian DNAmethyltransferases: a route to new mechanisms. EMBO Rep. 12, 647–656. doi:10.1038/embor.2011.11

Desplats, P., Spencer, B., Coffee, E., Patel, P., Michael, S., Patrick, C., et al. (2011).Alpha-synuclein sequesters Dnmt1 from the nucleus: a novel mechanism forepigenetic alterations in Lewy body diseases. J. Biol. Chem. 286, 9031–9037. doi:10.1074/jbc.C110.212589

Dhayalan, A., Rajavelu, A., Rathert, P., Tamas, R., Jurkowska, R. Z., Ragozin, S.,et al. (2010). The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethy-lation and guides DNA methylation. J. Biol. Chem. 285, 26114–26120. doi:10.1074/jbc.M109.089433

Dhe-Paganon, S., Syeda, F., and Park, L. (2011). DNA methyl transferase 1: regu-latory mechanisms and implications in health and disease. Int. J. Biochem. Mol.Biol. 2, 58–66.

Ding, F., and Chaillet, J. R. (2002). In vivo stabilization of the Dnmt1 (cytosine-5)-methyltransferase protein. Proc. Natl. Acad. Sci. U.S.A. 99, 14861–14866. doi:10.1073/pnas.232565599

Dodge, J. E., Okano, M., Dick, F., Tsujimoto, N., Chen, T., Wang, S., et al.(2005). Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNAhypomethylation, chromosomal instability, and spontaneous immortalization.J. Biol. Chem. 280, 17986–17991. doi: 10.1074/jbc.M413246200

Dong, A., Yoder, J. A., Zhang, X., Zhou, L., Bestor, T. H., and Cheng, X. (2001).Structure of human DNMT2, an enigmatic DNA methyltransferase homologthat displays denaturant-resistant binding to DNA. Nucleic Acids Res. 29,439–448. doi: 10.1093/nar/29.2.439

Dzitoyeva, S., Chen, H., and Manev, H. (2012). Effect of aging on 5-hydroxymethylcytosine in brain mitochondria. Neurobiol. Aging 33, 2881–2891. doi: 10.1016/j.neurobiolaging.2012.02.006






Easwaran, H. P., Schermelleh, L., Leonhardt, H., and Cardoso, M. C. (2008).Replication-independent chromatin loading of Dnmt1 during G2 and Mphases. EMBO Rep. 5, 1181–1186. doi: 10.1038/sj.embor.7400295

Esteller, M. (2008). Epigenetics in cancer. N. Engl. J. Med. 358, 1148–1159. doi:10.1056/NEJMra072067

Farge, G., Mehmedovic, M., Baclayon, M., van den Wildenberg, S. M., Roos,W. H., Gustafsson, C. M., et al. (2014). In vitro-reconstituted nucleoids canblock mitochondrial DNA replication and transcription. Cell Rep. 8, 66–74. doi:10.1016/j.celrep.2014.05.046

Fatemi, M., Hermann, A., Gowher, H., and Jeltsch, A. (2002). Dnmt3a and Dnmt1functionally cooperate during de novo methylation of DNA. Eur. J. Biochem.269, 4981–4984. doi: 10.1046/j.1432-1033.2002.03198.x

Feinberg, A. P. (2007). Phenotypic plasticity and the epigenetics of human disease.Nature 447, 433–440. doi: 10.1038/nature05919

Fellinger, K., Rothbauer, U., Felle, M., Längst, G., and Leonhardt, H. (2009).Dimerization of DNAmethyltransferase 1 ismediated by its regulatory domain.J. Cell. Biochem. 106, 521–528. doi: 10.1002/jcb.22071

Fontana, A., Gast, H., Reith, W., Recher, M., Birchler, T., and Bassetti, C. L. (2010).Narcolepsy: autoimmunity, effector T cell activation due to infection, or T cellindependent, major histocompatibility complex class II induced neuronal loss?Brain 133, 1300–1311. doi: 10.1093/brain/awq086

Forbes, S. A., Bhamra, G., Bamford, S., Dawson, E., Kok, C., Clements, J., et al.(2008). The catalogue of somatic mutations in cancer (COSMIC). Curr. Protoc.Hum. Genet. 10, 11. doi: 10.1002/0471142905.hg1011s57

Ghosh, S., Sengupta, S., and Scaria, V. (2014). Comparative analysis of humanmitochondrial methylomes shows distinct patterns of epigenetic regulation inmitochondria.Mitochondrion 18, 58–62. doi: 10.1016/j.mito.2014.07.007

Goll, M. G., Kirpekar, F., Maggert, K. A., Yoder, J. A., Hsieh, C. L., Zhang, X.,et al. (2006). Methylation of tRNAAsp by the DNA methyltransferase homologDnmt2. Science 311, 395–398. doi: 10.1126/science.1120976

Goyal, R., Reinhardt, R., and Jeltsch, A. (2006). Accuracy of DNA methylationpattern preservation by the Dnmt1 methyltransferase. Nucl. Acids Res. 34,1182–1188. doi: 10.1093/nar/gkl002

Guo, J. U., Su, Y., Shin, J. H., Shin, J., Li, H., Xie, B., et al. (2014). Distribution,recognition, and regulation of non-CpG methylation in the adult mammalianbrain. Nat. Neurosci. 17, 215–222. doi: 10.1038/nn.3607

Hata, K., Okano, M., Lei, H., and Li, E. (2002). Dnmt3L cooperates with the Dnmt3family of de novo DNA methyltransferases to establish maternal imprints inmice. Development 129, 1983–1993.

Hermann, A., Goyal, R., and Jeltsch, A. (2004). The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preferencefor hemimethylated target sites. J. Biol. Chem. 279, 48350–48359. doi:10.1074/jbc.M403427200

Hermann, A., Schmitt, S., and Jeltsch, A. (2003). The human Dnmt2 has residualDNA-(cytosine-C5)methyltransferase activity. J. Biol. Chem. 278, 31717–31721.doi: 10.1074/jbc.M305448200

Hojo, K., Imamura, T., Takanashi, M., Ishii, K., Sasaki, M., Imura, S., et al.(1999). Hereditary sensory neuropathy with deafness and dementia: a clini-cal and neuroimaging study. Eur. J. Neurol. 6, 357–361. doi: 10.1046/j.1468-1331.1999.630357.x

Holliday, R., and Pugh, J. E. (1975). DNAmodification mechanisms and gene activ-ity during development. Science 187, 226–232. doi: 10.1126/science.1111098

Hong, E. E., Okitsu, C. Y., Smith, A. D., and Hsieh, C. L. (2013). Regionally spe-cific and genome-wide analyses conclusively demonstrate the absence of CpGmethylation in human mitochondrial DNA.Mol. Cell. Biol. 33, 2683–2690. doi:10.1128/MCB.00220-13

Hor, H., Bartesaghi, L., Kutalik, Z., Vicário, J. L., de Andrés, C., Pfister, C., et al.(2011). A missense mutation in myelin oligodendrocyte glycoprotein as a causeof familial narcolepsy with cataplexy. Am. J. Hum. Genet. 89, 474–479. doi:10.1016/j.ajhg.2011.08.007

Howard, G., Eiges, R., Gaudet, F., Jaenisch, R., and Eden, A. (2008). Activation andtransposition of endogenous retroviral elements in hypomethylation inducedtumors in mice. Oncogene 27, 404–408. doi: 10.1038/sj.onc.1210631

Iacobazzi, V., Castegna, A., Infantino, V., and Andria, G. (2013). MitochondrialDNA methylation as a next-generation biomarker and diagnostic tool. Mol.Genet. Metab. 110, 25–34. doi: 10.1016/j.ymgme.2013.07.012

Iacobuzio-Donahue, C. A. (2009). Epigenetic changes in cancer. Annu. Rev. Pathol.4, 229–249. doi: 10.1146/annurev.pathol.3.121806.151442

Infantino, V., Castegna, A., Iacobazzi, F., Spera, I., Scala, I., Andria, G., et al.(2011). Impairment of methyl cycle affects mitochondrial methyl availabilityand glutathione level in Down’s syndrome. Mol. Genet. Metab. 102, 378–382.doi: 10.1016/j.ymgme.2010.11.166

Ishida, M. I., and Moore, G. E. (2013). The role of imprinted genes in humans.Mol.Aspects Med. 34, 826–840. doi: 10.1016/j.mam.2012.06.009

Jakovcevski, M., and Akbarian, S. (2012). Epigenetic mechanisms in neurode-velopmental and neurodegenerative disease. Nat. Med. 18, 1194–1204. doi:10.1038/nm.2828

Jeltsch, A. (2002). Beyond Watson and Crick: DNA methylation and molecu-lar enzymology of DNA methyltransferases. Chembiochem 3, 274–293. doi:10.1002/1439-7633(20020402)3:4<274::AID-CBIC274>3.0.CO;2-S

Jeltsch, A. (2006). Molecular enzymology of mammalian DNAmethyltransferases.Curr. Top Microbiol. Immunol. 301, 203–225. doi: 10.1007/3-540-31390-7_7

Jeltsch, A., and Jurkowska, R. Z. (2014). New concepts in DNAmethylation. TrendsBiochem. Sci. 39, 310–318. doi: 10.1016/j.tibs.2014.05.002

Jia, D., Jurkowska, R. Z., Zhang, X., Jeltsch, A., and Cheng, X. (2007). Structureof Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation.Nature 449, 248–251. doi: 10.1038/nature06146

Jones, P. A., and Baylin, S. B. (2007). The epigenomics of cancer. Cell 128, 683–692.doi: 10.1016/j.cell.2007.01.029

Jones, P. A., and Liang, G. (2009). Rethinking how DNA methylation patterns aremaintained. Nat. Rev. Genet. 10, 805–811. doi: 10.1038/nrg2651

Jowaed, A., Schmitt, I., Kaut, O., and Wüllner, U. (2010). Methylation reg-ulates alpha-synuclein expression and is decreased in Parkinson’s diseasepatients’ brains. J. Neurosci. 30, 6355–6359. doi: 10.1523/JNEUROSCI.6119-09.2010

Jurkowska, R. Z., Jurkowski, T. P., and Jeltsch, A. (2011a). Structure and func-tion of mammalian DNA methyltransferases. Chembiochem 12, 206–222. doi:10.1002/cbic.201000195

Jurkowska, R. Z., Rajavelu, A., Anspach, N., Urbanke, C., Jankevicius, G., Ragozin,S., et al. (2011b). Oligomerization and binding of the Dnmt3a DNA methyl-transferase to parallel DNAmolecules: heterochromatic localization and role ofDnmt3L. J. Biol. Chem. 286, 24200–24207. doi: 10.1074/jbc.M111.254987

Jurkowski, T. P., Meusburger, M., Phalke, S., Helm, M., Nellen,W., Reuter, G., et al. (2008). Human DNMT2 methylatestRNA(Asp) molecules using a DNA methyltransferase-like cat-alytic mechanism. RNA 14, 1663–1670. doi: 10.1261/rna.970408

Kaneda, M., Okano, M., Hata, K., Sado, T., Tsujimoto, N., Li, E., et al. (2004).Essential role for de novo DNA methyltransferase Dnmt3a in paternal andmaternal imprinting. Nature 429, 900–903. doi: 10.1038/nature02633

Kanherkar, R. R., Bhatia-Dey, N., and Csoka, A. B. (2014a). Epigenetics across thehuman lifespan. Front. Cell Dev. Biol. 2:49. doi: 10.3389/fcell.2014.00049

Kanherkar, R. R., Bhatia-Dey, N., Makarev, E., and Csoka, A. B. (2014b). Cellularreprogramming for understanding and treating human disease. Front. Cell Dev.Biol. 2:67. doi: 10.3389/fcell.2014.00067

Kar, S., Deb, M., Sengupra, D., Shilpi, A., Parbin, S., Torrisani, J., et al.(2012). An insight into the various regulatory mechanisms modulating humanDNA methyltransferase 1 stability and function. Epigenetics 9, 994–1007. doi:10.4161/epi.21568

Kelly, R. D., Mahmud, A., McKenzie, M., Trounce, I. A., and St John, J. C. (2012).Mitochondrial DNA copy number is regulated in a tissue specific manner byDNA methylation of the nuclear-encoded DNA polymerase gamma A. NucleicAcids Res. 40, 10124–10138. doi: 10.1093/nar/gks770

Kimura, F., Seifert, H. H., Florl, A. R., Santourlidis, S., Steinhoff, C., Swiatkowski,S., et al. (2003). Decrease of DNA methyltransferase 1 expression relative tocell proliferation in transitional cell carcinoma. Int. J. Cancer 104, 568–578. doi:10.1002/ijc.10988

Klein, C. J., Bird, T., Ertekin-Taner, N., Lincoln, S., Hjorth, R., Wu Y., et al. (2013).DNMT1 mutation hot spot causes varied phenotypes of HSAN1 with demen-tia and hearing loss. Neurology 80, 824–828. doi: 10.1212/WNL.0b013e318284076d

Klein, C. J., Botuyan, M. V., Wu, Y., Ward, C. J., Nicholson, G. A., Hammans, S.,et al. (2011). Mutations in DNMT1 cause hereditary sensory neuropathy withdementia and hearing loss.Nat. Genet. 43, 595–600. doi: 10.1038/ng.830

Kohli, R. M., and Zhang, Y. (2013). TET enzymes, TDG and the dynamics of DNAdemethylation. Nature 502, 472–479. doi: 10.1038/nature12750






Kondilis-Mangum, H. D., and Wade, P. A. (2013). Epigenetics andthe adaptive immune response. Mol. Aspects Med. 34, 813–825. doi:10.1016/j.mam.2012.06.008

Kornum, B. R., Kawashima, M., Faraco, J., Lin, L., Rico, T. J., Hesselson, S., et al.(2011). Common variants in P2RY11 are associated with narcolepsy.Nat. Genet.43, 66–71. doi: 10.1038/ng.734

Krishnakumar, R., and Blelloch, R. H. (2013). Epigenetics of cellular repro-gramming. Curr. Opin. Genet. Dev. 23, 548–555. doi: 10.1016/j.gde.2013.06.005

Landan, G., Cohen, N. M., Mukamel, Z., Bar, A., Molchadsky, A., Brosh, R., et al.(2012). Epigenetic polymorphism and the stochastic formation of differentiallymethylated regions in normal and cancerous tissues.Nat. Genet. 44, 1207–1214.doi: 10.1038/ng.2442

Lande-Diner, L., Zhang, J., Ben-Porath, I., Amariglio, N., Keshet, I., Hecht, M., et al.(2007). Role of DNA methylation in stable gene repression. J. Biol. Chem. 282,12194–12200. doi: 10.1074/jbc.M607838200

Lascaro, D., Castellana, S., Gasparre, G., Romeo, G., Saccone, C., and Attimonelli,M. (2008). The RHNumtS compilation: features and bioinformatics approachesto locate and quantify HumanNumtS. BMCGenomics 9:267. doi: 10.1186/1471-2164-9-267

Lashley, T., Gami, P., Valizadeh, N., Li, A., Revesz, T., and Balazs, R.(2014). Alterations in global DNA methylation and hydroxymethylationare not detected in Alzheimer’s disease. Neuropathol. Appl. Neurobiol. doi:10.1111/nan.12183 [Epub ahead of print].

Laurent, L., Wong, E., Li, G., Huynh, T., Tsirigos, A., Ong, C. T., et al. (2010).Dynamic changes in the human methylome during differentiation.Genome Res.20, 320–331. doi: 10.1101/gr.101907.109

Lee, P. P., Fitzpatrick, D. R., Beard, C., Jessup, H. K., Lehar, S., Makar, K. W.,et al. (2001). A critical role for Dnmt1 and DNA methylation in T cell devel-opment, function, and survival. Immunity 15, 763–774. doi: 10.1016/S1074-7613(01)00227-8

Lee, T., Zhai, J., and Meyers, B. C. (2010). Conservation and divergence in eukary-otic DNA methylation. Proc. Natl. Acad. Sci. U.S.A. 107, 9027–9028. doi:10.1073/pnas.1005440107

Leonhardt, H., Page, A. W., Weier, H. U., and Bestor, T. H. (1992). A tar-geting sequence directs DNA methyltransferase to sites of DNA replica-tion in mammalian nuclei. Cell 71, 865–873. doi: 10.1016/0092-8674(92)90561-P

Li, E., Bestor, T. H., and Jaenisch, R. (1992). Targeted mutation of the DNAmethyltransferase gene results in embryonic lethality. Cell 69, 915–926. doi:10.1016/0092-8674(92)90611-F

Li, G., Zan, H., Xu, Z., and Casali, P. (2013). Epigenetics of the antibody response.Trends Immunol. 34, 460–470. doi: 10.1016/j.it.2013.03.006

Loughery, J. E., Dunne, P. D., O’Neill, K. M., Meehan, R. R., McDaid, J. R.,and Walsh, C. P. (2011). DNMT1 deficiency triggers mismatch repair defectsin human cells through depletion of repair protein levels in a processinvolving the DNA damage response. Hum. Mol. Genet. 20, 3241–3255. doi:10.1093/hmg/ddr236

Lu, H., Liu, X., Deng, Y., and Qing, H. (2013). DNA methylation, ahand behind neurodegenerative diseases. Front. Aging Neurosci. 5:85. doi:10.3389/fnagi.2013.00085

Maekawa, M., Taniguchi, T., Higashi, H., Sugimura, H., Sugano, K., and Kanno, T.(2004). Methylation of mitochondrial DNA is not a useful marker for cancerdetection. Clin. Chem. 50, 1480–1481. doi: 10.1373/clinchem.2004.035139

Mastroeni, D., McKee, A., Grover, A., Rogers, J., and Coleman, P. D. (2009).Epigenetic differences in cortical neurons from a pair of monozygotic twinsdiscordant for Alzheimer’s disease. PLoS ONE 4:e6617. doi: 10.1371/jour-nal.pone.0006617

Matsumoto, L., Takuma, H., Tamaoka, A., Kurisaki, H., Date, H., Tsuji, S., et al.(2010). CpG demethylation enhances alpha-synuclein expression and affectsthe pathogenesis of Parkinson’s disease. PLoS ONE 5:e15522. doi: 10.1371/jour-nal.pone.0015522

Melberg, A., Dahl, N., Hetta, J., Valind, S., Nennesmo, I., Lundberg, P.O., and Raininko, R. (1999). Neuroimaging study in autosomal dominantcerebellar ataxia, deafness, and narcolepsy. Neurology 53, 2190–2192. doi:10.1212/WNL.53.9.2190

Melberg, A., Hetta, J., Dahl, N., Nennesmo, I., Bengtsson, M., Wibom, R.,et al. (1995). Autosomal dominant cerebellar ataxia deafness and narcolepsy.J. Neurol. Sci. 134, 119–129. doi: 10.1016/0022-510X(95)00228-0

Moghadam, K. K., Pizza, F., La Morgia, C., Franceschini, C., Tonon, C., Lodi, R.,et al. (2014). Narcolepsy is a common phenotype in HSAN IE and ADCA-DN.Brain 137, 1643–1655. doi: 10.1093/brain/awu069

Mortusewicz, O., Schermelleh, L., Walter, J., Cardoso, M. C., and Leonhardt, H.(2005). Recruitment of DNAmethyltransferase I to DNA repair sites. Proc. Natl.Acad. Sci. U.S.A. 102, 8905–8909. doi: 10.1073/pnas.0501034102

Nass, M. M. (1973). Differential methylation of mitochondrial and nuclear DNAin cultured mouse, hamster and virus-transformed hamster cells. In vivo and invitro methylation. J. Mol. Biol. 80, 155–175. doi: 10.1016/0022-2836(73)90239-8

Okano, M., Bell, D. W., Haber, D. A., and Li, E. (1999). DNA methyltransferasesDnmt3a and Dnmt3b are essential for de novo methylation and mammaliandevelopment. Cell 99, 247–257. doi: 10.1016/S0092-8674(00)81656-6

Okano, M., Xie, S., and Li, E. (1998). Cloning and characterization of a fam-ily of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. 19,219–220. doi: 10.1038/890

O’Keefe, R. T., Henderson, S. C., and Spector, D. L. (1992). Dynamic organizationof DNA replication in mammalian cell nuclei: spatially and temporally definedreplication of chromosome-specific alpha-satellite DNA sequences. J. Cell. Biol.116, 1095–1110. doi: 10.1083/jcb.116.5.1095

Ooi, S. K., Qiu, C., Bernstein, E., Li, K., Jia, D., Yang, Z., et al. (2007). DNMT3Lconnects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.Nature 448, 714–717. doi: 10.1038/nature05987

Otani, J., Nankumo, T., Arita, K., Inamoto, S., Ariyoshi, M., and Shirakawa, M.(2009). Structural basis for recognition of H3K4 methylation status by theDNAmethyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep. 10,1235–1241. doi: 10.1038/embor.2009.218

Papp, B., and Plath, K. (2013). Epigenetics of reprogramming to induced pluripo-tency. Cell 152, 1324–1343. doi: 10.1016/j.cell.2013.02.043

Pedroso, J. L., Povoas Barsottini, O. G., Lin, L., Melberg, A., Oliveira, A. S., andMignot, E. (2013). A novel de novo exon 21 DNMT1mutation causes cerebellarataxia, deafness, and narcolepsy in a Brazilian patient. Sleep 36, 1257–1259.

Peyron, C., Faraco, J., Rogers,W., Ripley, B., Overeem, S., Charnay, Y., et al. (2000).A mutation in a case of early onset narcolepsy and a generalized absence ofhypocretin peptides in human narcoleptic brains. Nat. Med. 6, 991–997. doi:10.1038/79690

Pfeifer, G. P., Tang, M., and Denissenko,M. F. (2000). Mutation hotspots and DNAmethylation. Curr. TopMicrobiol. Immunol. 249, 1–19. doi: 10.1007/978-3-642-59696-4_1

Pirola, C. J., Gianotti, T. F., Burgueño, A. L., Rey-Funes, M., Loidl, C. F., Mallardi,P., et al. (2013). Epigenetic modification of liver mitochondrial DNA is asso-ciated with histological severity of nonalcoholic fatty liver disease. Gut 62,1356–1363. doi: 10.1136/gutjnl-2012-302962

Pollack, Y., Kasir, J., Shemer, R., Metzger, S., and Szyf, M. (1984). Methylationpattern of mouse mitochondrial DNA. Nucleic Acids Res. 12, 4811–4824. doi:10.1093/nar/12.12.4811

Pradhan, M., Estève, P. O., Chin, H. G., Samaranayke, M., Kim, G. D., and Pradhan,S. (2008). CXXC domain of human DNMT1 is essential for enzymatic activity.Biochemistry 47, 10000–100009. doi: 10.1021/bi8011725

Qin, W., Leonhardt, H., and Pichler, G. (2011). Regulation of DNA methyl-transferase 1 by interactions and modifications. Nucleus 2, 392–402. doi:10.4161/nucl.2.5.17928

Qiu, C., Sawada, K., Zhang, X., and Cheng, X. (2002). The PWWP domain of mam-malian DNA methyltransferase Dnmt3b defines a new family of DNA-bindingfolds. Nat. Struct. Biol. 9, 217–224.

Qureshi, I. A., and Mehler, M. F. (2013). Epigenetic mechanisms govern-ing the process of neurodegeneration. Mol. Aspects Med. 34, 875–882. doi:10.1016/j.mam.2012.06.011

Rao, J. S., Keleshian, V. L., Klein, S., and Rapoport, S. I. (2012). Epigenetic modifi-cations in frontal cortex from Alzheimer’s disease and bipolar disorder patients.Transl. Psychiatry 2:e132. doi: 10.1038/tp.2012.55

Rebelo, A. P., Williams, S. L., and Moraes, C. T. (2009). In vivo methylation ofmtDNA reveals the dynamics of protein-mtDNA interactions. Nucleic AcidsRes. 37, 6701–6715. doi: 10.1093/nar/gkp727






Reik, W. (2007). Stability and flexibility of epigenetic gene regulation inmammalian development. Nature 447, 425–432. doi: 10.1038/nature05918

Riggs, A. D. (1975). X inactivation, differentiation, and DNA methylation.Cytogenet. Cell Genet.14, 9–25. doi: 10.1159/000130315

Rivera, C. M., and Ren, B. (2013). Mapping human epigenomes. Cell 155, 39–55.doi: 10.1016/j.cell.2013.09.011

Robertson, K. D., Uzvolgyi, E., Liang, G., Talmadge, C., Sumegi, J., Gonzales, F. A.,et al. (1999). The human DNA methyltransferases (DNMTs) 1, 3a and 3b:coordinate mRNA expression in normal tissues and overexpression in tumors.Nucleic Acids Res. 27, 2291–2298. doi: 10.1093/nar/27.11.2291

Rountree, M. R., Bachman, K. E., and Baylin, S. B. (2000). DNMT1 binds HDAC2and a new co-repressor, DMAP1, to form a complex at replication foci. Nat.Genet. 25, 269–277. doi: 10.1038/77023

Schaefer, M., and Lyko, F. (2010). Solving the Dnmt2 enigma. Chromosoma 119,35–40. doi: 10.1007/s00412-009-0240-6

Schaefer, M., Steringer, J. P., and Lyko, F. (2008). The Drosophila cytosine-5 methyltransferase Dnmt2 is associated with the nuclear matrix and canaccess DNA during mitosis. PLoS ONE 3:e1414. doi: 10.1371/journal.pone.0001414

Schon, E. A., and Przedborski, S. (2011). Mitochondria: the next (neurode) gener-ation. Neuron 70, 1033–1053. doi: 10.1016/j.neuron.2011.06.003

Sha, K. Y. (2008). A mechanistic view of genomic imprinting. Annu. Rev. Genom.Hum. Genet. 9, 197–216. doi: 10.1146/annurev.genom.122007.110031

Shmookler, R. J., and Goldstein, S. (1983). Mitochondrial DNA in mortal andimmortal human cells. Genome number, integrity, and methylation. J. Biol.Chem. 258, 9078–9085.

Shock, L. S., Thakkar, P. V., Peterson, E. J., Moran, R. G., and Taylor, S. M. (2011).DNAmethyltransferase 1, cytosine methylation, and cytosine hydroxymethyla-tion in mammalian mitochondria. Proc. Natl. Acad. Sci. U.S.A. 108, 3630–3635.doi: 10.1073/pnas.1012311108

Song, J., Rechkoblit, O., Bestor, T. H., and Patel, D. J. (2011). Structure ofDNMT1-DNA complex reveals a role for autoinhibition in maintenance DNAmethylation. Science 331, 1036–1040. doi: 10.1126/science.1195380

Straub, T., and Becker, P. B. (2007). Dosage compensation: the beginning and endof generalization. Nat. Rev. Genet. 8, 47–57. doi: 10.1038/nrg2013

Subramaniam, D., Thombre, R., Dhar, A., and Anant, S. (2014). DNA methyl-transferases: a novel target for prevention and therapy. Front. Oncol. 4:80. doi:10.3389/fonc.2014.00080

Sun, Z., Wu, Y., Ordog, T., Baheti, S., Nie, J., Duan, X., et al. (2014). Aberrantsignature methylome by DNMT1 hot spot mutation in hereditary sensory andautonomic neuropathy 1E. Epigenetics 9, 1184–1193. doi: 10.4161/epi.29676

Sung, H. Y., Choi, E. N., AhnJo, S., Oh, S., and Ahn, J. H. (2011). Amyloid pro-tein mediated differential DNA methylation status regulates gene expressionin Alzheimer’s disease model cell line. Biochem. Biophys. Res. Commun. 414,700–705. doi: 10.1016/j.bbrc.2011.09.136

Syeda, F., Fagan, R. L., Wean, M., Avvakumov, G. V., Walker, J. R., Xue, S.,et al. (2011). The replication focus targeting sequence (RFTS) domain is aDNA-competitive inhibitor of Dnmt1. J. Biol. Chem. 286, 15344–15351. doi:10.1074/jbc.M110.209882

Tafti, M., Hor, H., Dauvilliers, Y., Lammers, G. J., Overeem, S., Mayer, G., et al.(2014). DQB1 locus alone explains most of the risk and protection in narcolepsywith cataplexy in Europe. Sleep 37, 19–25.

Takai, D., and Jones, P. A. (2004). Origins of bidirectional promoters: computa-tional analyses of intergenic distance in the human genome.Mol. Biol. Evol. 21,463–467. doi: 10.1093/molbev/msh040

Takeshita, K., Suetake, I., Yamashita, E., Suga, M., Narita, H., Nakagawa, A.,et al. (2011). Structural insight into maintenance methylation by mouse DNAmethyltransferase 1 (Dnmt1). Proc. Natl. Acad. Sci. U.S.A. 108, 9055–9059. doi:10.1073/pnas.1019629108

Tatton-Brown, K., Seal, S., Ruark, E., Harmer, J., Ramsay, E., Del Vecchio Duarte,S., et al. (2014). Mutations in the DNAmethyltransferase gene DNMT3A causean overgrowth syndrome with intellectual disability. Nat. Genet. 46, 385–388.doi: 10.1038/ng.2917

Valenti, D., Manente, G. A., Moro, L., Marra, E., and Vacca, R. A. (2011). Deficitof complex I activity in human skin fibroblasts with chromosome 21 trisomyand overproduction of reactive oxygen species by mitochondria: involve-ment of the cAMP/PKA signalling pathway. Biochem. J. 435, 679–688. doi:10.1042/BJ20101908

Vanyushin, B. F., and Kirnos, M. D. (1974). The nucleotide composition andpyrimidine clusters in DNA from beef heart mitochondria. FEBS Lett. 39,195–199. doi: 10.1016/0014-5793(74)80049-9

Vanyushin, B. F., and Kirnos, M. D. (1977). Structure of animal mitochon-drial DNA (base composition, pyrimidine clusters, character of methylation).Biochim. Biophys. Acta 475, 323–336. doi: 10.1016/0005-2787(77)90023-5

Vanyushin, B. F., Kiryanov, G. I., Kudryashova, I. B., and Belozersky, A. N. (1971).DNA-methylase in loach embryos (Misgurnus fossilis). FEBS Lett. 15, 313–316.doi: 10.1016/0014-5793(71)80646-4

Vilkaitis, G., Suetake, I., Klimasauskas, S., and Tajima, S. (2005). Processive methy-lation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase.J. Biol. Chem. 280, 64–72. doi: 10.1074/jbc.M411126200

Wang, L., Liu, Y., Beier, U. H., Han, R., Bhatti, T. R., Akimova, T., et al.(2013). Foxp3+ T-regulatory cells require DNA methyltransferase 1 expres-sion to prevent development of lethal autoimmunity. Blood 121, 3631–3639.doi: 10.1182/blood-2012-08-451765

Weemaes, C. M., van Tol, M. J., Wang, J., van Ostaijen-ten Dam, M. M., vanEggermond, M. C., Thijssen, P. E., et al. (2013). Heterogeneous clinical presen-tation in ICF syndrome: correlation with underlying gene defects. Eur. J. Hum.Genet. 21, 1219–1225. doi: 10.1038/ejhg.2013.40

Weissman, J., Naidu, S., and Bjornsson, H. T. (2014). Abnormalities of the DNAmethylation mark and its machinery: an emerging cause of neurologic dysfunc-tion. Semin. Neurol. 34, 249–257. doi: 10.1055/s-0034-1386763

Wijsman, E. M., Daw, E.W., Yu, C. E., Payami, H., Steinbart, E. J., Nochlin, D., et al.(2004). Evidence for a novel late-onset Alzheimer disease locus on chromosome19p13.2. Am. J. Hum. Genet. 75, 398–409. doi: 10.1086/423393

Winkelmann, J., Lin, L., Schormair, B., Kornum, B. R., Faraco, J., Plazzi, G.,et al. (2012). Mutations in DNMT1 cause autosomal dominant cerebel-lar ataxia, deafness and narcolepsy. Hum. Mol. Genet. 21, 2205–2210. doi:10.1093/hmg/dds035

Wong, M., Gertz, B., Chestnut, B. A., and Martin, L. J. (2013). MitochondrialDNMT3A and DNA methylation in skeletal muscle and CNS of transgenicmousemodels of ALS. Front. Cell Neurosci. 7:279. doi: 10.3389/fncel.2013.00279

Wu, B. K., Mei, S. C., and Brenner, C. (2014). RFTS-deleted DNMT1 enhancestumorigenicity with focal hypermethylation and global hypomethylation. CellCycle 13, 3222–3231. doi: 10.4161/15384101.2014.950886

Wu, H., Zeng, H., Lam, R., Tempel, W., Amaya, M. F., Xu, C., et al. (2011).Structural and histone binding ability characterizations of human PWWPdomains. PLoS ONE 6:e18919. doi: 10.1371/journal.pone.0018919

Wu, H., and Zhang, Y. (2014). Reversing DNA methylation: mech-anisms, genomics, and biological functions. Cell 156, 45–68. doi:10.1016/j.cell.2013.12.019

Xu, G. L., Bestor, T. H., Bourc’his, D., Hsieh, C. L., Tommerup, N., Bugge, M.,et al. (1999). Chromosome instability and immunodeficiency syndrome causedby mutations in a DNA methyltransferase gene. Nature 402, 187–191. doi:10.1038/46214

Yu, L., Chibnik, L. B., Srivastava, G. P., Pochet, N., Yang, J., Xu, J., et al.(2014). Association of Brain DNAMethylation in SORL1, ABCA7, HLA-DRB5,SLC24A4, and BIN1 With Pathological Diagnosis of Alzheimer Disease. JAMANeurol. 72, 15–24. doi: 10.1001/jamaneurol.2014.3049

Yuan, J., Higuchi, Y., Nagado, T., Nozuma, S., Nakamura, T., Matsuura, E., et al.(2013). Novel mutation in the replication focus targeting sequence domain ofDNMT1 causes hereditary sensory and autonomic neuropathy IE. J. Peripher.Nerv. Syst. 18, 89–93. doi: 10.1111/jns5.12012

Zhang, Y., Jurkowska, R., Soeroes, S., Rajavelu, A., Dhayalan, A., Bock, I., et al.(2010). Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guidedby interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res.38, 4246–53. doi: 10.1093/nar/gkq147

Conflict of Interest Statement: The authors declare that the research was con-ducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

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